CN111944851A - Nanoscale siRNA delivery system - Google Patents
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- CN111944851A CN111944851A CN201910407443.5A CN201910407443A CN111944851A CN 111944851 A CN111944851 A CN 111944851A CN 201910407443 A CN201910407443 A CN 201910407443A CN 111944851 A CN111944851 A CN 111944851A
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Abstract
The invention discloses a nanoscale siRNA delivery system, wherein a star-shaped oligo-arginine carrier loads siRNA to form a stable binary compound, and then the nanoscale siRNA delivery system with the function of self-accelerating endosome escape is prepared by coating weak acid sensitive polyanion. The system can carry out intelligent disassembly in an acidic endosome/lysosome environment, and regenerate the original binary compound and PLL, and the escape of the endosome/lysosome of siRNA can be accelerated in situ in the process. In addition, the system also has good serum stability and biocompatibility. Based on the advantages, the system shows better delivery efficiency compared with a binary compound, and effectively inhibits the migration of vascular smooth muscle cells.
Description
Technical Field
The invention belongs to the technical field of gene carrier materials, and particularly relates to a nanoscale siRNA delivery system with a self-accelerating endosome escape function, and a preparation method and application thereof.
Background
Abnormal proliferation and migration of vascular smooth muscle cells are important causes of intimal hyperplasia in blood vessels, and provide effective targets for prevention and treatment of intimal hyperplasia. In recent years, emerging genetic engineering techniques, particularly RNA interference techniques, have achieved satisfactory results in the diagnosis and treatment of diseases (e.g., cancer, cardiovascular diseases, etc.). Thus, we believe that silencing target mrnas, proteins, and related cellular pathways in vascular smooth muscle cells using siRNA delivery technology would be an effective strategy to inhibit vascular smooth muscle cell proliferation and migration. However, the bottleneck that has been limiting the therapeutic efficacy of siRNA is the lack of safe, efficient vectors. Therefore, the development of multifunctional siRNA delivery carriers makes it critical to deliver siRNA to vascular smooth muscle cells safely and efficiently.
In recent years, the rise of cell penetrating peptide research has injected new activities into the design of vectors in the field of gene/drug delivery. Generally, researchers will load genes and exert their function of penetrating cell membranes by cationic cell penetrating peptides, such as oligoarginine, thereby delivering genes into cells with high efficiency. However, this strategy also faces problems of non-specific serum adsorption and low endosome/lysosome escape. In order to further solve the problems, researchers find that the polyanion coating technology can effectively reduce serum adsorption in the blood circulation process, increase the serum stability of the gene complex and prolong the blood circulation time. However, it fails to simultaneously achieve the function of enhancing endosomal/lysosomal escape. Recent developments in smart polymer materials have provided a new field of view for the design of the above-described polymer-based carriers for drug/gene delivery, especially responses triggered by weak acid environments. The prior art can support chemical synthesis of a weak acid sensitive high molecular material which has the property of keeping stable and presenting polyanion under neutral or weakly alkaline conditions, but the property of triggering hydrolysis reaction of specific amido bond to reduce polycation when the material is placed in a weak acid environment. This process can be triggered within the acidic endosome/lysosomal organelle within the cell.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a nanoscale siRNA delivery system with a function of self-accelerating endosome escape, and a preparation method and application thereof.
A nanometer siRNA delivery system and a preparation method thereof are carried out according to the following steps:
Diallyl functionalized cage polysilsesquioxane (POSS- (DA)8) And oligo-arginine W-G-R8Uniformly dispersing the-G-C in a mixed solution of tetrahydrofuran and water, adding a photoinitiator, uniformly mixing, reacting under the irradiation of an ultraviolet lamp, dialyzing after the reaction is finished, and freeze-drying to obtain star-type oligoarginine POSS- (C-G-R)8-G-W)16The mass ratio of the powder, the diallyl functionalized cage polysilsesquioxane and the oligoarginine is 2: (30-60);
in step 1, POSS is an abbreviation for cage polysilsesquioxane, POSS- (DA)8Is diallyl functionalized cage type polysilsesquioxane, oligoarginine W-G-R8-the amino acid sequence of G-C is: Trp-Gly-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Gly-Cys.
In step 1, the volume ratio of tetrahydrofuran to water is (2: 3) - (1: 2).
In step 1, the mass ratio of diallyl functionalized cage polysilsesquioxane to oligoarginine is 2: (40-50).
In the step 1, after the reaction is finished, the reaction solution is placed in a dialysis bag with the cut-off molecular weight of 2000-8000Da (number average molecular weight) and is dialyzed for 48 to 72 hours by using distilled water, and freeze drying is carried out to obtain the star-shaped oligo-arginine POSS- (C-G-R)8-G-W)16And (3) powder.
In the step 1, the reaction is carried out for 10-15min under the irradiation of an ultraviolet lamp.
In step 1, the photoinitiator is 2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone, and the addition amount is 0.05-0.5%, preferably 0.1-0.5% of the sum of the masses of the diallyl functionalized cage type polysilsesquioxane, the oligomeric arginine and the photoinitiator.
Uniformly dispersing polylysine in a PBS solution, cooling to 0 ℃, adding cis-aconitic anhydride powder, adding a sodium hydroxide aqueous solution to maintain the overall pH at 8-9, and continuously stirring for reaction to obtain cis-aconitic anhydride modified polylysine;
in the step 2, the mass ratio of polylysine to cis-aconitic anhydride is 1: (1-5), preferably 1: (1.5-4.5).
In step 2, the reaction is carried out in PBS at a concentration of 10mM, pH 8.5, polylysine number average molecular weight 4200Da and stirring for 12-24 hours.
In step 2, after the reaction is finished, the reaction solution is placed in a dialysis bag with the molecular weight cut-off of 500-. PLL is an abbreviation for polylysine, PLL-g-Aco is an abbreviation for cis-aconitic anhydride modified polylysine.
Uniformly dispersing the star-shaped oligoarginine powder prepared in the step 1 in a PBS (phosphate buffer solution) solution to obtain a first solution; adding siRNA into the first solution, wherein the mass ratio of the star-shaped oligoarginine to the siRNA is (0.5-6): 1, vortexing and incubating at room temperature to obtain a binary siRNA compound solution;
uniformly dispersing the weak acid sensitive polyanion prepared in the step 2 in a PBS (phosphate buffer solution) solution to obtain a second solution; adding a second solution into the binary siRNA compound solution, wherein the mass ratio of the star-shaped oligoarginine to the weak acid-sensitive polyanion is 6: (1-5), vortexing and incubating at room temperature to obtain a ternary siRNA complex solution which is used as a nanoscale siRNA delivery system with the function of self-accelerating endosome escape.
In step 3, the PBS solution was at pH7.4 and 10 mM.
In step 3, the star-shaped oligoarginine powder is uniformly dispersed in the PBS solution, vortexed for 2min to obtain a first solution, then siRNA is added, vortexed for 1min, and incubated at room temperature for 20-60min to obtain a binary siRNA compound solution.
In step 3, the mass ratio of the star-shaped oligoarginine to the siRNA is (2-6): 1.
in step 3, the mass ratio of the star-shaped oligoarginine to the weak acid-sensitive polyanion is 6: (2-3).
In step 3, the second solution is added to the binary siRNA complex solution, vortexed for 1min, and incubated at room temperature for 20-60 min.
In step 3, the weak acid sensitive polyanion is uniformly dispersed in the PBS solution, and vortexed for 2min to obtain a second solution with the mass concentration of 0.1-0.3 mg/mL.
In step 3, the siRNA is ERK2-siRNA or Cy5-ERK 2-siRNA.
In the present invention, the room temperature is 20 to 30 ℃.
The application of the nano-scale siRNA delivery system in cell transfection embodies the function of self-accelerating endosome escape, such as accelerating the endosome/lysosome escape efficiency of siRNA, efficiently inhibiting the migration of vascular smooth muscle cells and inhibiting vascular intimal hyperplasia.
In the invention, cationic cell penetrating peptide oligoarginine is covalently coupled to an active arm on cage polysilsesquioxane to prepare the star-shaped oligoarginine gene vector. The carrier can load siRNA through electrostatic interaction to form a stable binary siRNA compound. In order to improve the serum stability of the binary complex and prolong the blood circulation time of the binary complex, polyanion sensitive to weak acid environment is electrostatically coated on the surface of the binary complex to form a ternary complex, which is also called as an siRNA delivery system. The delivery system has good serum stability and can be efficiently taken up by vascular smooth muscle cells. In addition, when the siRNA is trapped in an endosome/lysosome environment, the weak acid-sensitive polyanion can be gradually triggered and reduced into polycation, and then the polycation is separated from the surface of the binary complex under the induction of electrostatic repulsion, and the original binary complex and the polycation are regenerated, so that the endosome/lysosome escape efficiency of the siRNA can be remarkably accelerated, and the delivery efficiency and the silencing effect of the siRNA are promoted.
Compared with the prior art, the star-shaped oligoarginine carrier prepared by the invention has stronger siRNA loading capacity, and the formed siRNA compound has good stability. Solves the problems of difficult loading, easy degradation and the like of siRNA in the traditional sense. The nano-scale siRNA delivery system with the function of self-accelerating endosome escape can be intelligently disassembled in an acidic endosome/lysosome environment, and can regenerate the original binary compound and-PLL, and the process can accelerate the endosome/lysosome escape of siRNA in situ. In addition, the system also has good serum stability and biocompatibility. Based on the advantages, compared with the binary siRNA compound, the system has better delivery efficiency and silencing effect, and effectively inhibits the migration of vascular smooth muscle cells.
Drawings
FIG. 1 shows POSS- (C-G-R) in the present invention8-G-W)16Nuclear magnetic resonance hydrogen spectrum of (a).
FIG. 2 shows NMR spectra of PLL-g-Aco and PLL-g-Suc according to the present invention.
FIG. 3 is an agarose gel electrophoresis of different mass ratios of binary siRNA complexes and delivery systems.
FIG. 4 is a graph of particle size and Zeta potential for different mass ratios of siRNA delivery systems.
FIG. 5 is a graph showing the results of the siRNA delivery material and the cytotoxicity test of the system according to the present invention.
FIG. 6 is a graph of the results of Zeta potential change monitoring weak acid induced siRNA delivery system disassembly behavior.
FIG. 7 is a graph of experimental results of endosome/lysosome escape effects and efficiency of siRNA.
Fig. 8 is a graph showing the experimental results of the transwell migration experiment of vascular smooth muscle cells.
Detailed Description
The present invention will be described in further detail with reference to the following examples. In the present example, oligoarginine W-G-R8-the amino acid sequence of G-C is: Trp-Gly-Arg-Arg-Arg-Arg-Arg-Arg-Gly-Cys, available from Gill Biochemical (Shanghai) Co., Ltd. PLL is available from Shanghai-derived leaf Biotechnology, Inc. POSS- (DA)8Is diallyl functionalized cage polysilsesquioxane and is synthesized in a laboratory. ERK2-siRNA was purchased from Yangzhou Ruibo Biotechnology Ltd and has the nucleotide sequence: forward: 5'-GATCCGCACCTCAGCAATGATCATCTTCCTGTCAGAATGATCATTGCTGAGGTGCTTTTTG-3', respectively; 5'-AATTCAAAAAGCACCTCAGCAATGATCATTCTGACAGGAAGATGATCATTGCTGAGGTGCG-3' for Reverse.
Example 1: star-shaped oligo-arginine POSS- (C-G-R)8-G-W)16Preparation of Gene vector
2mgPOSS- (DA)8And 43mg of W-G-R8dissolving-G-C in 4.5mL of tetrahydrofuran/water mixed solution (volume ratio is 1/2), adding 1mg of photoinitiator 2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone, uniformly mixing, irradiating the mixture under an ultraviolet lamp for 10min to react, putting the reaction solution into a dialysis bag with molecular weight cutoff of 3500Da, dialyzing the reaction solution with distilled water for 72h, and freeze-drying to obtain star-shaped oligo-arginine POSS- (C-G-R)8-G-W)16And (3) powder. According to the nuclear magnetic resonance detection, as shown in figure 1, no characteristic signal peak of allyl is found in the range of chemical shift 5-6ppm in the spectrum, and W-G-R is observed at 7-8ppm8Characteristic signal peaks of the indole group in-G-C, which may indicate complete allylic substitution by W-G-R during the reaction8The (G) -C addition to produce the target product POSS- (C-G-R)8-G-W)16。
Example 2: preparation of star-shaped oligo-arginine carrier/ERK 2-siRNA binary complex
5mg of POSS- (C-G-R) product of example 1 were taken8-G-W)16Dissolve in 10mL PBS (10mM, pH 7.4) and vortex for 2min to give solution one. According to POSS- (C-G-R)8-G-W)16The mass ratio of the carrier to the ERK2-siRNA is 0.5:1, 1:1, 2:1, 3:1, 4:1, 5:1 and 6:1 respectively, the ERK2-siRNA solution is added into the first solution, vortex for 30s, and incubation is carried out for 30min at room temperature, so as to obtain the star-shaped oligo-arginine carrier/ERK 2-siRNA binary complex.
Example 3: preparation of nanoscale siRNA delivery system with 'self-accelerating endosome escape' function
(1) Preparation of weak acid sensitive polyanion:
50mg of commercial-PLL (number average molecular weight 4200Da) was dissolved in 15mL of PBS (10mM, pH 8.5) to obtain an aqueous solution of-PLL. Cooling the solution to 0 ℃, slowly adding 122mg of cis-aconitic anhydride powder into the solution, continuously supplementing 1M sodium hydroxide aqueous solution into the reaction solution in the process, keeping the pH value of the reaction solution at 8.5, continuously stirring for 12h, placing the reaction solution into a dialysis bag with the cut-off molecular weight of 1000Da, dialyzing for 72h by using distilled water with the pH value of 8-9, and freeze-drying to obtain the weak acid sensitive polyanion PLL-g-Aco powder.
(2) 2mg of the product PLL-g-Aco powder obtained in step (1) was weighed out and dissolved in 10mL of PBS (10mM, pH 7.4) to obtain a PLL-g-Aco solution. Adding a PLL-G-Aco solution into the binary siRNA compound solution prepared in the embodiment 2 with the mass ratio of 6:1, vortexing for 1min, and incubating at room temperature for 30min to obtain the nano-scale siRNA delivery system with the function of self-accelerating endosome escape, wherein the POSS- (C-G-R)8-G-W)16The mass ratio of ERK2-siRNA to PLL-g-Aco is 6:1: (1-5).
Example 4: preparation of non-weak acid sensitive polyanion coated nano-scale siRNA delivery system
(1) Preparation of non-weak acid sensitive polyanion:
50mg of commercial-PLL (number average molecular weight 4200Da) was dissolved in 15mL of PBS (10mM, pH 8.5) to obtain an aqueous solution of-PLL. Cooling the solution to 0 ℃, slowly adding 78mg of succinic anhydride powder into the solution, continuously supplementing 1M sodium hydroxide aqueous solution into the reaction solution in the process, keeping the pH value of the reaction solution at 8.5, continuously stirring the reaction solution for 12 hours, placing the reaction solution into a dialysis bag with the molecular weight cut-off of 1000Da, dialyzing the reaction solution for 72 hours by using distilled water, and freeze-drying the dialysis solution to obtain the non-weak acid sensitive polyanion PLL-g-Suc powder. PLL is an abbreviation for polylysine, PLL-g-Suc is an abbreviation for succinic anhydride modified polylysine.
(2) 2mg of the product PLL-g-Suc powder obtained in step (1) was weighed out and dissolved in 10mL of PBS (10mM, pH 7.4) to obtain a PLL-g-Suc solution. Into example 2Adding a PLL-G-Suc solution into the prepared binary siRNA compound solution with the mass ratio of 6:1, whirling for 1min, and incubating at room temperature for 30min to obtain the non-weak acid sensitive polyanion coated nanoscale siRNA delivery system, wherein the POSS- (C-G-R) is8-G-W)16The mass ratio of ERK2-siRNA to PLL-g-Suc is 6:1: 3.
NMR examination was performed on PLL-g-Aco and PLL-g-Suc prepared in examples 3 and 4, as shown in FIG. 2, and characteristic peaks in the main structure of PLL were labeled in FIG. 2B, in which characteristic peaks h and i of A succinamide moiety were around 2.5ppm and characteristic peaks f and g of B cis aconitamide moiety were around 3.2ppm and 5.9ppm, respectively, indicating the chemical structures of PLL-g-Suc and PLL-g-Aco.
Example 5: preparation of star-shaped oligo-arginine vector/Cy 5-ERK2-siRNA binary complex
Solution one of example 2 was prepared and was prepared according to POSS- (C-G-R)8-G-W)16And the mass ratio of the Cy-5-ERK 2-siRNA to the Cy-5-ERK 2-siRNA is 6:1, adding the Cy-5-ERK 2-siRNA solution into the first solution, whirling for 30s, and incubating for 30min at room temperature to obtain a star-shaped oligo-arginine carrier/Cy-5-ERK 2-siRNA binary complex solution. Cy5-ERK2-siRNA is an abbreviation for Cy5 labeled ERK 2-siRNA.
Example 6: preparation of nanoscale Cy5-siRNA delivery System with "self-accelerating endosome escape" function
Separately preparing the PLL-G-Aco solution in example 3 and the binary complex solution in example 5, adding the PLL-G-Aco solution to the binary complex solution, vortexing for 1min, and incubating at room temperature for 30min to obtain a nanoscale Cy5-siRNA delivery system with a function of self-accelerating endosome escape, wherein the POSS- (C-G-R) is8-G-W)16The mass ratio of Cy5-ERK2-siRNA to PLL-g-Aco is 6:1: 3.
Example 7: preparation of non-weak acid sensitive polyanion coated nanoscale Cy5-siRNA delivery system
Respectively preparing the PLL-g-Suc solution in example 4 and the binary compound solution in example 5, adding the PLL-g-Suc solution into the binary compound solution, vortexing for 1min, and incubating at room temperature for 30min to obtain the non-weak acid sensitive polyanion coated nanoscale Cy5siRNA delivery System, the POSS- (C-G-R)8-G-W)16The mass ratio of Cy5-ERK2-siRNA to PLL-g-Suc is 6:1: 3.
Example 8: ability and stability of nano-scale siRNA delivery system to load ERK2-siRNA
Sample solutions in example 2 and example 3 in mass ratios of 0.5:1, 1:1, 2:1, 3:1, 4:1 were prepared, as were sample solutions in example 3 in mass ratios of 6:1:1, 6:1:3, 6:1: 5. The sample solution is mixed with 6 × loading buffer, then electrophoresis is carried out for 30min under the conditions of 1 × TAE buffer solution, 0.8% agarose gel and 100V voltage, and then the distribution position of ERK2-siRNA in gel electrophoresis is observed under the irradiation of an ultraviolet lamp and recorded by photographing. The results are shown in FIG. 3, where a and b are the results of binary complex and siRNA delivery system, respectively, and columns 1 and 2 in the a and b graphs indicate marker and ERK2-siRNA, respectively, the latter column indicates the mass ratio, and a is POSS- (C-G-R)8-G-W)16The mass ratio of the siRNA to the ERK2-siRNA is that b is POSS- (C-G-R)8-G-W)16Mass ratio to ERK2-siRNA to PLL-g-Suc.
The experimental result shows that POSS- (C-G-R)8-G-W)16When the mass ratio of the ERK2-siRNA to the ERK2-siRNA is 2:1, the ERK2-siRNA is completely loaded. And, on the basis of the binary complex with the mass ratio of 6:1, the stability of the delivery system is not affected by the addition of the weak acid-sensitive polyanion.
Example 9: particle size and Zeta potential of the nanoscale siRNA delivery system.
Sample solutions of example 2 and example 3 at 6:1, 6:1:3 and 6:1:5 mass ratios and the sample solution of example 4 were prepared and the particle size and Zeta potential of all the solutions were measured using a Zetasizer Nano ZS instrument with the results shown in fig. 4, the left side coordinate being the particle size and the right side coordinate being the Zeta potential, the leftmost test results (i.e., two in one set of histograms) corresponding to the abscissa being the sample solution of example 2 at 6:1 mass ratio, the rightmost test results being the sample solution of example 4, the middle three sets corresponding to example 3 at 6:1:1, 6:1:3 and 6:1:5 mass ratios, the left side histogram corresponding to the particle size and the right side histogram corresponding to the Zeta potential in each set of histograms. The experimental results show that the polyanion coated binary siRNA compound can increase the particle size of a delivery system and reduce the Zeta potential. Meanwhile, the prepared siRNA delivery system has the particle size value of 100-200nm and is suitable for cell transfection.
Example 10: cytotoxicity test
Rat arterial vascular smooth muscle cells were cultured at 8X 103The culture medium is replaced by serum-free DMEM medium, and the incubation is continued for 12 hours. Adding polymer materials with different concentrations and siRNA compound, mixing well, culturing for 4h, replacing with 10% FBS-containing culture medium, and culturing for 48 h. Thereafter, 100. mu.L of serum-free medium containing 0.5mg/mL MTT was added to each well, the culture was continued for 4 hours, the culture medium was discarded, 100. mu.L of DMSO was added, and shaking was carried out for 10 min. The microplate reader measures the Optical Density (OD) value at a wavelength of 490 nm. The relative cell activity value (%) can be calculated by the following formula, and the results are shown in FIG. 5, in which ERK2-siRNA and POSS- (C-G-R) are shown in the order from left to right in the histogram (30/50/100/200/400nM) of siRNA concentration of each group8-G-W)16、POSS-(C-G-R8-G-W)16/Scr siRNA、POSS-(C-G-R8-G-W)16/ERK2-siRNA、POSS-(C-G-R8-G-W)16/Scr siRNA/PLL-g-Aco、POSS-(C-G-R8-G-W)16ERK2-siRNA/PLL-G-Aco, where Scr-siRNA is an abbreviation for inactive siRNA, solution one in example 2 was prepared and followed by POSS- (C-G-R)8-G-W)16Adding the Scr-siRNA solution into the first solution, whirling for 30s, and incubating at room temperature for 30min to obtain star-shaped oligo-arginine carrier/Scr-siRNA binary compound solution, namely POSS- (C-G-R)8-G-W)16Scr siRNA; then adding the PLL-G-Aco solution in the example 3 into the star-shaped oligo-arginine carrier/Scr-siRNA binary compound solution, vortexing for 1min, and incubating at room temperature for 30min to obtain a nanoscale Scr-siRNA delivery system with a self-accelerating endosome escape function, wherein the POSS- (C-G-R)8-G-W)16The mass ratio of the Scr-siRNA to the PLL-G-Aco is 6:1:3, namely POSS- (C-G-R)8-G-W)16/Scr siRNA/PLL-g-Aco。
Relative cellular activity (%) ═ OD490nm(Experimental group)/OD490nm(control group) × 100%
The experimental results show that: in the experimental concentration range, the relative cell activity value is higher than 80%, which indicates that the siRNA delivery system has good cell compatibility.
Example 11: zeta potential change monitoring weak acid induction system disassembly behavior
The sample solutions of example 3 and example 4 were taken in a mass ratio of 6:1:3, adjusted to pH7.4 and 5.5 with 0.1M hydrochloric acid solution, and Zeta potential values of 1h, 2h, 4h, 7h, 10h, and 13h were recorded using a Zetasizer Nano ZS instrument, and the results are shown in FIG. 6, where 1 is the Zeta potential value at pH5.5 for the sample solution of example 3 in a mass ratio of 6:1:3, 2 is the Zeta potential value at pH7.4 for the sample solution of example 3 in a mass ratio of 6:1:3, 3 is the Zeta potential value at pH5.5 for the sample solution of example 4, and 4 is the Zeta potential value at pH7.4 for the sample solution of example 4.
The experimental results show that: at pH7.4, no significant change in Zeta potential occurred in both sets of sample solutions. Under the weak acid environment with the pH value of 5.5, the Zeta potential value of the delivery system prepared by coating the weak acid sensitive polyanion gradually increases from the weak negative potential to the original binary compound level with time, and the Zeta potential value of the delivery system prepared by coating the non-weak acid sensitive polyanion is still not obviously changed. This may indicate the process: weak acids can induce the disassembly of sensitive delivery systems, allowing the regeneration of the original binary siRNA complex and-PLL.
Example 12: experiment of endosome/lysosome escape efficiency of siRNA.
Rat arterial vascular smooth muscle cells were cultured at 8X 104The cells were inoculated into a confocal plate and cultured for 24 hours, and then transfected with the sample solution of example 5, the sample solution of example 6 in a mass ratio of 6:1:3, and the sample solution of example 7 in a serum-free medium for 4 hours, respectively, and cultured for 24 hours again in a DMEM medium containing 10% FBS. The cells were washed twice with PBS, incubated for 1h with a pre-warmed solution containing 0.5mM Lyso Tracker Green, and washed with pre-warmed PBS2 times, further incubation with 2. mu.g/mL of Hoechst 33342 solution for 20min in the greenhouse, washing the cells twice with PBS, and finally observing Cy5-ERK2-siRNA (red dots) and lysosomes (green dots) and nuclei (blue dots), respectively, with confocal laser microscopy under excitation at 645nm, 504nm and 350 nm. Wherein: the red dot + green dot is yellow dot, and the red dot + blue dot is pink dot.
The co-localization ratio of Cy5-ERK2-siRNA in lysosomes was calculated using the following formula.
The co-location ratio (100%) [ yellow dot number)/(red dot number + yellow dot number + pink dot number) ] x 100%
As a result, as shown in FIG. 7, BRCs were the sample solution of example 5, TRCs-Aco were the sample solution of example 6 at a mass ratio of 6:1:3, and TRCs-Suc were the sample solution of example 7, and in each set of the bar graphs, the left side was 4 hours, and the right side was 24 hours. The experimental results show that: at the initial 4h, the binary complex exhibited higher efficiency of endosome/lysosome escape. In the time period of 4h-24h, intelligent disassembly and assembly of the weak acid sensitive delivery system occur, the escape efficiency of siRNA is greatly accelerated, and the siRNA is finally superior to that of binary compounds, and the process is actually self-accelerated inclusion/lysosome escape in situ.
Example 13: cell migration assay
Rat arterial vascular smooth muscle cells were transfected in 24-well plates using the sample solutions in example 2 at a mass ratio of 6:1, example 3 at a mass ratio of 6:1:3, and example 4 for 24h, centrifuged, and plated at 1X 105The density of cells/well was re-dispersed into the upper chamber of the transwell, cultured for 6h, the migrated cells were labeled with crystal violet, photographed and the cell mobility calculated by Image-Pro Plus 6.0 software, and the results are shown in fig. 8, where a is ERK2-siRNA (negative control group), B is commercial transfection reagent Lipofectamine 3000 (positive control group), C is the sample solution of example 2 at a mass ratio of 6:1, D is the sample solution of example 3 at a mass ratio of 6:1:3, and E is the sample solution of example 4. The experimental result shows that compared with a binary compound and a non-weak acid sensitive delivery system, the nanoscale siRNA delivery system with the function of self-accelerating endosome escape has better effect of inhibiting smooth muscle cell migrationThe effect of the shift.
The preparation of siRNA delivery systems can be achieved by adjusting the process parameters according to the teachings of the present invention, and exhibit substantially consistent performance with the present invention. The invention has been described in an illustrative manner, and it is to be understood that any simple variations, modifications or other equivalent changes which can be made by one skilled in the art without departing from the spirit of the invention fall within the scope of the invention.
Claims (10)
1. A nano-scale siRNA delivery system is characterized in that the particle size is 100-200nm, and the delivery system is carried out according to the following steps:
step 1, star-type oligomeric arginine POSS- (C-G-R)8-G-W)16Preparation of Gene vector
Diallyl functionalized cage type polysilsesquioxane and oligoarginine W-G-R8Uniformly dispersing the-G-C in a mixed solution of tetrahydrofuran and water, adding a photoinitiator, uniformly mixing, reacting under the irradiation of an ultraviolet lamp, dialyzing after the reaction is finished, and freeze-drying to obtain star-type oligoarginine POSS- (C-G-R)8-G-W)16The mass ratio of the powder, the diallyl functionalized cage polysilsesquioxane and the oligoarginine is 2: (30-60);
step 2, preparation of weak acid sensitive polyanion PLL-g-Aco
Uniformly dispersing polylysine in a PBS solution, cooling to 0 ℃, adding cis-aconitic anhydride powder, adding a sodium hydroxide aqueous solution to maintain the overall pH at 8-9, and continuously stirring for reaction to obtain cis-aconitic anhydride modified polylysine;
step 3, preparation of nano-scale siRNA delivery system
Uniformly dispersing the star-shaped oligoarginine powder prepared in the step 1 in a PBS (phosphate buffer solution) solution to obtain a first solution; adding siRNA into the first solution, wherein the mass ratio of the star-shaped oligoarginine to the siRNA is (0.5-6): 1, vortexing and incubating at room temperature to obtain a binary siRNA compound solution;
uniformly dispersing the weak acid sensitive polyanion prepared in the step 2 in a PBS (phosphate buffer solution) solution to obtain a second solution; adding a second solution into the binary siRNA compound solution, wherein the mass ratio of the star-shaped oligoarginine to the weak acid-sensitive polyanion is 6: (1-5), vortexing and incubating at room temperature to obtain a ternary siRNA complex solution, namely a nanoscale siRNA delivery system.
2. The delivery system of claim 1, wherein in step 1, the oligo-arginine W-G-R8-the amino acid sequence of G-C is: Trp-Gly-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Gly-Cys; the volume ratio of tetrahydrofuran to water is (2: 3) - (1: 2); the mass ratio of the diallyl functionalized cage-type polysilsesquioxane to the oligoarginine is 2: (40-50); the reaction is carried out for 10-15min under the irradiation of an ultraviolet lamp, the photoinitiator is 2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone, and the addition amount is 0.05% -0.5%, preferably 0.1-0.5% of the sum of the masses of the diallyl functionalized cage type polysilsesquioxane, the oligomeric arginine and the photoinitiator.
3. The nano-scale siRNA delivery system according to claim 1, wherein in step 2, the mass ratio of-polylysine to cis-aconitic anhydride is 1: (1-5), preferably 1: (1.5-4.5); in PBS, 10mM, pH 8.5, number average molecular weight of polylysine is 4200Da, and the reaction time is 12-24 hours with stirring.
4. The nano-scale siRNA delivery system according to claim 1, wherein in step 3, the siRNA is ERK2-siRNA or Cy5-ERK 2-siRNA; the pH of the PBS solution is 7.4, and the concentration is 10 mM; the mass ratio of the star-shaped oligoarginine to the siRNA is (2-6): 1; the mass ratio of the star-shaped oligoarginine to the weak acid-sensitive polyanion is 6: (2-3).
5. The nano-scale siRNA delivery system according to claim 1, wherein in step 3, star-shaped oligo-arginine powder is uniformly dispersed in PBS solution, vortexed for 2min to obtain a first solution, then siRNA is added, vortexed for 1min, and incubated at room temperature for 20-60min to obtain a binary siRNA complex solution; adding the second solution into the binary siRNA compound solution, vortexing for 1min, and incubating at room temperature for 20-60 min; and uniformly dispersing the weak acid sensitive polyanion in the PBS solution, and vortexing for 2min to obtain a second solution with the mass concentration of 0.1-0.3 mg/mL.
6. A preparation method of a nano-scale siRNA delivery system is characterized by comprising the following steps:
step 1, star-type oligomeric arginine POSS- (C-G-R)8-G-W)16Preparation of Gene vector
Diallyl functionalized cage type polysilsesquioxane and oligoarginine W-G-R8Uniformly dispersing the-G-C in a mixed solution of tetrahydrofuran and water, adding a photoinitiator, uniformly mixing, reacting under the irradiation of an ultraviolet lamp, dialyzing after the reaction is finished, and freeze-drying to obtain star-type oligoarginine POSS- (C-G-R)8-G-W)16The mass ratio of the powder, the diallyl functionalized cage polysilsesquioxane and the oligoarginine is 2: (30-60);
step 2, preparation of weak acid sensitive polyanion PLL-g-Aco
Uniformly dispersing polylysine in a PBS solution, cooling to 0 ℃, adding cis-aconitic anhydride powder, adding a sodium hydroxide aqueous solution to maintain the overall pH at 8-9, and continuously stirring for reaction to obtain cis-aconitic anhydride modified polylysine;
step 3, preparation of nano-scale siRNA delivery system
Uniformly dispersing the star-shaped oligoarginine powder prepared in the step 1 in a PBS (phosphate buffer solution) solution to obtain a first solution; adding siRNA into the first solution, wherein the mass ratio of the star-shaped oligoarginine to the siRNA is (0.5-6): 1, vortexing and incubating at room temperature to obtain a binary siRNA compound solution;
uniformly dispersing the weak acid sensitive polyanion prepared in the step 2 in a PBS (phosphate buffer solution) solution to obtain a second solution; adding a second solution into the binary siRNA compound solution, wherein the mass ratio of the star-shaped oligoarginine to the weak acid-sensitive polyanion is 6: (1-5), vortexing and incubating at room temperature to obtain a ternary siRNA complex solution, namely a nanoscale siRNA delivery system.
7. The method of claim 6, wherein in step 1, the oligo-arginine W-G-R is selected from the group consisting of L-arginine, L8-the amino acid sequence of G-C is: Trp-Gly-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Gly-Cys; the volume ratio of tetrahydrofuran to water is (2: 3) - (1: 2); the mass ratio of the diallyl functionalized cage-type polysilsesquioxane to the oligoarginine is 2: (40-50); the reaction is carried out for 10-15min under the irradiation of an ultraviolet lamp, the photoinitiator is 2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone, and the addition amount is 0.05% -0.5%, preferably 0.1-0.5% of the sum of the masses of the diallyl functionalized cage type polysilsesquioxane, the oligomeric arginine and the photoinitiator.
8. The method for preparing a nano-scale siRNA delivery system according to claim 6, wherein in step 2, the mass ratio of-polylysine to cis-aconitic anhydride is 1: (1-5), preferably 1: (1.5-4.5); in PBS, 10mM, pH 8.5, number average molecular weight of polylysine is 4200Da, and the reaction time is 12-24 hours with stirring.
9. The method for preparing a nano-scale siRNA delivery system according to claim 6, wherein in step 3, the siRNA is ERK2-siRNA or Cy5-ERK 2-siRNA; the pH of the PBS solution is 7.4, and the concentration is 10 mM; the mass ratio of the star-shaped oligoarginine to the siRNA is (2-6): 1; the mass ratio of the star-shaped oligoarginine to the weak acid-sensitive polyanion is 6: (2-3); uniformly dispersing star-shaped oligoarginine powder in a PBS (phosphate buffer solution), vortexing for 2min to obtain a first solution, adding siRNA, vortexing for 1min, and incubating at room temperature for 20-60min to obtain a binary siRNA compound solution; adding the second solution into the binary siRNA compound solution, vortexing for 1min, and incubating at room temperature for 20-60 min; and uniformly dispersing the weak acid sensitive polyanion in the PBS solution, and vortexing for 2min to obtain a second solution with the mass concentration of 0.1-0.3 mg/mL.
10. Use of a nanoscale siRNA delivery system according to any of claims 1 to 5 for cell transfection, wherein efficiency of endosome/lysosome escape of siRNA is accelerated, migration of vascular smooth muscle cells is inhibited with high efficiency, and intimal hyperplasia of blood vessels is inhibited.
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Application publication date: 20201117 |